World Journal
of Microbiology
and Biotechnology,
9, 256-264
Protein enrichment of sweet potato residue by solid-state cultivation with mono- and co-cultures of amylolytic fungi S.-S. Yang,* H.-D. Jang, C.-M. Liew and J.C. du Preez The degree of protein enrichment of sweet potato residue by different amylolytic moulds in solid-state cultivation was much greater than that obtained using amylolytic yeasts. The optimum initial moisture content for protein enrichment was about 65% (w/w). Adding nitrogen sources to the culture twice (at the start of the incubation and after 24 h) considerably improved the final protein content. A co-culture of amylolytic mycelial fungi yielded a at 30°C. In a column reactor, the highest product with 32% (w/w) crude protein after 4 days ’ incubation temperatures reached were 42°C and 40°C and the minimum 0, concentrations were 1.5% and 2.5% of full saturation in the central and bottom layers, respectively. Key words:
Amylolytic
fungi, co-culture,
protein
enrichment,
In 1990, Taiwan imported 1.99 x IO6 tons of soybeans, costing US$5.29 x 10’. from the USA (Economics and Planning Department 1991). Consequently, the development of local protein resources for animal feed from renewable raw materials is urgently required. The substrates traditionally used in solid-state cultivations for the production of foodstuffs are rice, wheat, millet, barley, maize and soybeans. However, use of agricultural wastes such as sweet potato residue, which is not naturally a good protein source for animal feed, might also be practicable in Taiwan because of their abundant supply and reasonable cost. Sweet potato starch is readily converted to biomass by many microorganisms capable of rapid growth (Yang & Chiu 1986; Yang 1988; Yang & Yuan 1990). In order to be economically viable, it must be possible to perform the bioconversion, of starchy materials into protein, at the rural level. Solid-state cultivation facilitates this by reducing the cost of microbial cultivation, by improving the in vitro rumen digestibility of the substrate, and by increasing the protein and fat contents of starchy or cellulosic materials (Grant et al. 1978; Raimbault & Alazard 1980; Yang 1988). We previously reported that
solid-state
cultivation.
sweet potato residue could be enriched from an initial value of 6% up to 21% (w/w) crude protein within 3 days by solid-state cultivation with amylolytic yeasts (Yang 1988). In this article, we report protein enrichment of sweet potato residue by solid-state cultivation with mono- and co-cultures of amylolytic moulds and yeasts, using static flask cultures as well as a column reactor.
Materials
and Methods
Sweet Potato Residue Sweet potato residue
was purchased from a local market in Taiwan and passed through a sieve (four to 16 mesh) to remove dust and large aggregates. The residue contained (w/w): 14.0 to 16.1% moisture; 2.32 to 3.11% crude protein; 2.7 to 3.6% ash; 16.1 to 18.0% crude fibre; and 65.4 to 70.0% carbohydrate (Yang 1988). Microorganisms Saccharomyces
cerevisiae Y-187, Y-188 and Y-191 were provided by C. F. Lin (Microbial Resources Institute, Taiwan). Saccharomyces diastaticLts IF0 1015 STA 1 (D), IF0 1046 STA 1, STA 2 (D) and Saccharomyces sp. IF0 1426 (D) were obtained from W. H. Wang (Research Institute for Wines, Taiwan Tobacco and Wine Monopoly
Bureau).
Schwanniomyces
occidentalis
853 was from
the
Department of Microbiology and Biochemistry, University of the Orange Free State, Bloemfontein, South Africa. Yarrowia (Candida] S.-S. Yang, H.-D. Jang and C.-M. Liew are with the Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan 10617, Republic of China; fax: 666 2 3633123. J.C. du Preez is with the Department of Microbiology and Biochemistry, University of the Orange Free State, Bloemfontein 9300, South Africa. * Corresponding author.
@ 1993 Rapid Communications
258
World Journal
of
of Oxford
Ltd
Microbiology and Biotechnology. Vol 9, 1993
Aspergillus niger Tainan, Mucor sp. NTLJ-AM-364 and Rhizoptrs sp. NRRL-688 and NRRL-695 were from the Department of Agricultural Chemistry, National Taiwan University, Taipei. All the microorganisms used had amylolytic activity.
lipolytica,
Protein C&m The
Media
and Cultlrre
spores or cells ml-’ with sterile water. For co-cultures a I:1 ratio of cells or spores was used. The solid substrate usually comprised (g): sweet potato residue, 100; (NH,),SO,, 1.25; urea, 1.25; and KH,PO,, 1.0. In some cases, different concentrations of the nitrogen sources were used (see Results). This substrate was distributed to Erlenmeyer flasks, autoclaved, mixed thoroughly with the spores or cells and the required amount of sterile water and incubated statically at 30°C for 3 to 9 days, with mixing once daily by rotating each flask by hand. The depth of medium in each flask was about 2 cm. The pH of the substrate was measured directly by immersing the electrode into the substrate, or determined after mixing an aliquot with five times its volume of distilled water. Coium~
Each sample for 20 min. directly by total nitrogen determined.
and Moisture
The
Fungal
Saccharomyces
1426 1046
of protein
amylolytic
for
further
fungi
enrichment
obtained in Table
study.
contents and pH of sweet for 4 days at 30°C.’
potato
residue
after
the growth
of different
Protein content (% w/w)
PH
Initial
Final
Initial
Final
Initial
Final
66 66
69 68
5.2 5.8
3.8 3.9
3.5
a.5
4.0 3.0
a.0 5.0
diastaticus
IF0
Sacc. Sacc.
cerevisiae cerevisiae
Y-187 Y-l 91
67 68
68 68
4.9 4.5
4.5 4.0
B53
66 66
74 76
5.6 5.3
3.0 3.2
3.0 3.5 4.0
5.5 10.0 11.0
67
78
4.6
3.7
3.5
la.0
66 65 66
72 71 69
4.7 4.5 4.2
4.1 4.1 4.0
3.5 3.5 3.0
15.0 16.5 13.5
A. niger Rhizopus Rhizopus Rhizopus * values per
Tainan sp. NRRL-688 sp. NRRL-695 sp. TBR are the means
g substrate
(dry
of triplicate wt).
experiments,
the
I. With
Sacc.
Schw. occidentalis Y. lipolytica
with
Rhizopcls and TBR, and A. niger Tainan,
is shown
Effect of Initial Moisture Content In all experiments, the moisture content of the substrate increased, the pH value decreased by varying degrees and protein enrichment was affected by initial moisture content. For example, with Saccharomyces sp. IF0 1426, the increase in moisture content was between 0.5% and 3.1% of total weight after 5 days (Table 2), whereas final protein content gradually increased with increasing initial moisture content up to 66.8% initial moisture. When the cultivation lasted more than 5 days, the protein content of the final product decreased slightly (data not shown). With Rhizopus sp.
Moisture content (% w/w)
sp. IF0
Organisms
sp. NRRL-688, NRRL-695 the protein content of the biomass was increased from an initial value of about 3% to 18% (w/w). Schw. occidenfalis B53 and Y. kpolytica gave final protein contents of 10% to ll%, and Sacc. cereuisiae Y-187 and Y-191, Sacc. diastaticus IF0 1046 and Saccharomyces sp. IF0 1426 gave 5% to 9%. In the light of these results, Saccharomyces sp. IF0 1426, Schw. occidentalis B53, Y. lipolyfica, A. niger Tainan and Rhizopus sp. NRRL-688, NRRL-695 and TBR were selected
Contents
strain
degree
different
was extracted with five vol distilled water by shaking and the soluble nitrogen in the extract was determined the Kjeldahl method (Meloan & Pomeranz 1980). The content of each sample, prior to extraction, was also The crude protein content was calculated from the
Table 1. The protein and moisture fungi in static solid-state culture
of Test
Selection
Reacior
Ash
potato residue
Results
The home-made, plastic column reactor (14.7 cm inner diam, 15.0 cm outer diam, 50.0 cm height) had equi-distant sampling holes, 10 cm apart along the column length, which were fitted with thermocouples and gas-sampling tubes. The temperature was measured with al-type thermocouple and digital thermometer. The 0, and CO, contents of the solid substrate were assayed by aspiration with a gas-tight syringe, sealed with a rubber bung, and analysis by gas chromatography using a thermal conductivity detector (Shimadzu GC-6A, Japan). Normal laboratory air was used as the control. No forced aeration was used. The solids were mixed by manually turning the column once daily.
Nitrogen, Protein,
of sweet
difference between the total nitrogen and soluble nitrogen contents, using the conversion factor of 6.25 (Yang et al. 1991). The ash content of samples was determined gravimetrically after 16 to 20 h at 550 to 600°C (Horwitz 1980). The moisture content of the culture was determined by drying a sample at 6O’C under vacuum for 8 to I2 h to constant mass (Yang 1988).
Conditions
amylolytic yeasts were cultivated at 30°C on yeast/malt extract/agar slants, containing (g I-‘): soluble starch, 10; yeast extract, 3; malt extract, 3; peptone, 5; and agar, 20. Moulds were cultivated at 30°C on potato/dextrose/agar slants, containing (g I-‘): potato infusion, 4; dextrose, 20; and agar, 20. Spores or cells were harvested with a Tween 80 solution (5 ml, 0.05% v/v) which was then adjusted to give lo7 to 10’
Solid-sfafe
enrichment
each
using
an inoculum
of IO’ to IO* spores
or cells
S.-S. Yung et al. Table 2. The effect of the residue In static solid-state
initial moisture content culture at 30°C.
on the protein
Parameter
Incubation 0
Saccharomyces sp. I FO 1426 Moisture content (% w/w)
.56
Protein
content
Rhizopus Moisture
(%
w/w)
sp. NRRL-695 content (% w/w)
PH
Protein
content
(% w/w)
56
75 80
75 80
-
81
-
-
-
-
4.5 4.9
-
-
-
-
4.9 4.7
-
-
-
3.2 3.3
3.4 3.7
3.2 3.2
4.9 5.2
3.2 3.2
5.6 5.6
56 60
56
65
60 66
69 76
71 76
80
81
-
-
3.6 5.3
-
6.9 6.5
-
6.4 6.2
-
57 61
57 63
67 71
68 73
76
78 82
81
potato
4
3
60 69 71 77
5.1 5.2
4.5 4.8
4.2 4.2
4.4 4.7
5.1 5.1
5.1 4.0
4.6
4.2 3.9
5.1 5.1
4.6 4.1
3.8 3.7 3.3
3.8 3.6
7.0 6.9
9.9 10.5
3.8 3.8
a.2 9.0 9.6 10.0
-
5
56 61 70 73 78 82
-
4.3 3.8
-
4.0
3.8 3.8 4.0 4.0
5.6 8.4 7.6 7.2 7.1
58 63 69 73 79 83 4.5 4.9 4.6
69 74 78 84 4.8 4.8 4.2
4.3
4.5 4.6 4.6
12.3 12.3
14.3 15.2
17.8 17.8
10.6 10.6 11.1
13.1 13.8 13.1
17.0 17.6
18.0 18.5
11.2
13.0
17.6 17.0
18.6 18.6
3.5 3.5
5.0 4.8
58 64
determined.
NRRL-695, the most rapid initial increase in protein content was found when initial moisture content exceeded 68.8% (Table 2) but by the end of the 5 days’ incubation protein contents were similar, irrespective of initial moisture content.
Nifrogen Supplemenfafion To improve the efficiency of utilization of the nitrogen source, a mixture of ammonium sulphate and urea (at a 1: 1
260
2
-
of sweet
(days)
56 59 67 71
3.3 3.4 -Not
time
59 67 71
4.8 4.6
PH
1
enrichment
World ]oumal of Microbiology and Biofechnology, Vol 9, 1993
w/w ratio) was added to the substrate at a rate of 2% (w/w) both at the start of the incubation (as usual) and again after 24 h. The addition of the nitrogen sources in two increments greatly enhanced the degree of protein enrichment, resulting in a final protein content 53% to 96% higher than in cultures not given extra nitrogen at 24 h. The highest protein contents were obtained with the moulds Rhizopus sp. NRRL-695 (26% w/w) and A. niger Tainan (29% w/w), whereas maximum protein contents obtained with the yeasts were only in the range of 17% to 18% (w/w) (Table 3).
Profein enrichment of sweet potafo residue
Table 3. The effect of nitrogen supplementation on the protein enrichment with different fungi in static flask solid-state culture for 4 days at 30°C. Fungai
strain
Moisture content (% w/w)
Saccharomyces sp. IF0 1426 Control’ Nitrogen supplementationt Sch w. occident&is 053 Control Nitrogen supplementation
of sweet
potato
residue
Protein content (% w/w)
PH
initial
Final
initial
Final
initial
Final
66 64
69 68
5.2 5.5
3.8 5.7
3.5 3.6
9 17
66 64
74 68
5.6 4.7
3.0 3.7
3.3 3.3
10 18
66 64
76 67
5.3 4.4
3.2 7.4
4.1 4.2
11 17
67 63
78 77
4.6 5.4
3.7 6.4
3.4 3.7
16 29
65 64
71 76
4.5 5.5
4.1 a.3
3.3 3.4
16 26
Y. lipolytica Control Nitrogen supplementation A. niger Tainan Control Nitrogen supplementation Rhizopus sp. NRRL-695 Control Nitrogen supplementation * Addition t Addition
of 1% (w/w) of 1% (w/w)
each each
of ammonium of ammonium
suiphate sulphate
Cultivation in a Column Reactor In the column reactor, there were increases in moisture content of between 1% and 4% and 8% and IO% total weight over 4 days with Saccharomyces sp. and A. niger, respectively. The medium became more acidic, changing from an initial pH of 4.3 to 4.4 to a final pH of 3.2 to 3.8. The Saccharomyces strain only gave a final mean protein content of 9% (w/w), whereas A. nip gave 19% (w/w).
Mono- and Co-cultures of Amylolytic Fungi Whereas the A. niger and l&ups sp. co-culture yielded the highest final protein content of the study (32% w/w), which was a slight improvement over the values obtained with these moulds in mono-cultures, the use of yeast co-cultures or a yeast together with a mould failed to improve on the final protein content obtained with the better of the two microorganisms in mono-culture (Table 4). Again, moisture content increased during incubation; the final moisture content was greater with the moulds than with the yeasts. Also as before, the variation in pH was largely dependent on the particular microorganism; some co-cultures became less acidic. For example, growth of Schw. occidentalis caused the pH to decrease to pH 3.6 within 2 days, whereas with Y. lipolytica it increased to pH 7.4, and with the moulds the pH value initially decreased and then increased. In the case of the co-cultures, the pattern of pH change depended on the combination of microorganisms used.
and urea and urea
prior prior
to inoculation. to inoculation
and again
after
24 h.
The ash content, which gives an indication of the amount of organic material lost through microbial metabolism (for example as CO,), increased during incubation; A. niger Tainan had the highest value (followed by the A. n&r Tainan and Rhizopus sp. NRRL-695 co-culture) and the Saccharomyces sp. IF0 1426 and Y. lipolytica co-culture had the lowest value. The observed increases in protein:ash ratios indicated that the increase in the protein content resulted from microbial growth and not only from the loss of organic material from the solids. Temperature and Oxygen ProjIes in &he Solid .%&rate The temperature profile in the solid substrate column reactor with Schw. occidentalis B53 is shown in Figure 1. The highest temperatures of 42°C and 40°C were reached in the central and bottom layers, respectively, after 18 h incubation at a room temperature of 23°C to 30°C. On mixing the solids, the temperatures decreased abruptly, then sharply increased during the subsequent active microbial growth which resulted from the increased 0, supply and fresh substrate contact. The temperatures of the central layer usually slightly exceeded those of the lower layer. The concentration of 0, in the solid substrate decreased gradually during incubation (Figure 2). Each time the column reactor was turned to mix the contents, the 0, concentration increased sharply, then decreased rapidly as vigorous microbial respiration proceeded. The lowest 0, concentrations reached were 1.5% and 2.5% in the bottom and central layers, respectively.
World Jamal
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S.-S. Yang et al.
Table 4. Protein enrichment of sweet potato residue cultivation at 30°C with nitrogen supplementation.” Fungal
Flask
strain
with mono-
Culture period (days)
and co-cultures
Moisture content (% w/w)
fungi
Schw.
sp. IF0
occidentalis
1426
853
PH
Ash (% w/w)
4.6 4.8
4 12
5.7 -
17 3
0
64
5.5
2 4
64
6.0 5.7
68 64
0 2
Tainan
Rhizopus
Protein content (% w/w)
Column
13 18
-
4 12
6.8 7.4
-
5.4 4.3
4.2 6.9
77 64
6.4 5.5
8.8 4.6
4
73 76
4.8 8.3
6.8 7.2
0
63
5.1
4.3
4
2 4
65
4.9 5.0
4.8
12
5.5 4.6
18 5
5.5 6.0 4.4
12 18 4
4.9 4.6 -
13
67 63 74
0 2 4 0 2
sp. NRRL-695
3.6 3.7 4.4
64 65
2 4
A. niger
4.7
69 68
4 0
Y. lipolytica
17 4 17 29 3 17 26
culture
Saccharomyces
Schw.
sp. IF0
occidentalis
Saccharomyces Y. lipolytica
1426
853
sp. IF0
SC/W. occidentalis Y. lipolytica Saccharomyces and Rhizopus
69 64
0 2 1426
and
853
and
sp. IF0
1426
sp. NRRL-695
4.8 4.5
4 0
89 71 65
2 4
66 66
5.9 7.6
0 2
64 69
4.5 5.1
4 0 2
71 65
6.3 5.5
4.6
70
4
73 64 73
4.3 4.2 4.7
5.9 6.4 -
4.4 5.5
0 2
4.6
-
Y. lipolytica
4 0 2
77 65
8.5 5.3
4.8
71
4
4.5 8.1
0 2
76 64 74
4 0 2 4
and
Rhizopus
sp. NRRL-695
A. niger Rhizopus
Tainan and sp. NRRL-695
Blank
l The culture conditions w/w) at 24 h.
were
as in Table
1, but with
19 4 13
-
Schw. occidentalis B53 and Rhizopus sp. NRRL-695
-Not
in a solid-state
culture
Saccharomyces
19 4 17 26 4 14
-
26 4
6.4
13
5.4 4.6
8.2 4.5 6.9
27 4 16
79 64 65
6.8 4.6 4.4
8.3 4.6 4.7
32 3
64
4.7
4.6
supplementation
with
(NH,),SOI
(1%
w/w)
3 3 and
urea
(1%
determined.
Discussion The water-holding capacity of a sample can be used as an index of aerobiosis (Wang 1981). The initial moisture content of the sweet potato residue used in these solid-state
262
of different
World Journal
of
Microbtology and Bmtechnology, Vof 9. 1993
cultivations was usually within the range of 64% to 68%; i.e. less than its water-holding capacity of 72% (Yang 1988). Therefore, suitable conditions for aerobic microbial growth existed in the cultures. During cultivation, however, the moisture content of the substrate increased substantially,
enrichment of sweet potato residue
Protein
bottom layer
201
^
R
40
20
0
Time Figure 2. of Schw. aspirated from the from the
22 0
20
40 Time (h)
60
80
100
Figure 1. Temperature profiles during the solid state cultivation of Schw. occidentalis 853 in a column reactor. Thermocouples were situated on the central vertical axis (A) or 3.5cm horizontally from it (0, a), either 10 cm from the bottom of the reactor or in the central layer, 20cm from the bottom of the reactor. A--Room temperature.
probably due to the production of metabolic water or the release of water resulting from the oxidation of carbohydrates. This increase has also been observed in mushroom spawn (Wang 1981), protein enrichment of sugar beet pulp (Durand & Chereau 1988), enzyme and antibiotic production from sweet potato residue (Yang 1992; Yang & Yuan 1990), liquor brewing from kuo-Lang (Lai 1984), and koji manufacture from cereals (Narahara et al. 1982). It follows, therefore, that the culture may become O,-limited as the cultivation proceeds, as indicated by Figure 2. That this can retard growth has been demonstrated in cultures of Schw. occidentalis 853, in which OJimitation severely restricted amylase production (Horn 1990) and in the inhibition of the degradation of organic materials in cornposting rubbish (Nakasaki et al. 1990). CO, formation and 0, consumption in solid-state fermentations are probably a function of radius position (Saucedo-Castaneda et al. 1990). Maximal CO, evolution rates were found in an intermediate region of the growth profile of A. tiger during protein enrichment of sugar-cane by-products (GonzalezBlanc0 et al. 1990). In the present study, 0, concentrations decreased as incubation progressed. Mixing or aeration may be necessary, therefore, for efficient protein enrichment by aerobic microbes in solid-state cultivation.
Oxygen
concentration
60
80
100
(h) during
the solid-state cultivation reactor. Gas samples were the centre of the horizontal plane either 1Ocm of the reactor (A) or in the central layer, 20 cm of the reactor (0).
occident&is 853 in a column from bottom bottom
The pH of solid-state cultivations can be controlled by using different ratios of ammonium salts and urea (Mitchell et al. 1988; Yang 1988), ammonium nitrate or sodium nitrate (Yang & Chiu 1986) a buffering agent such as CaCO,, or the salt of an organic acid (Reid 1989; Yang & Yuan 1990). However, our results show that use of a mixture of an ammonium salt and urea was only partially effective and that the degree of change in pH depended on the metabolic activities of the particular microorganism used. The large changes in pH noted in some of our cultivations can severely retard starch hydrolysis and growth. For example, the amylases of Schw. accidentalis B53 are rapidly and irreversibly inactivated below pH 5 (Horn 1990). In order to enrich the protein content of sweet potato residue to 20 to 30% (w/w), the addition of 4% (w/w) of a I: 1 mix of ammonium sulphate and urea was necessary. We found that the incremental supplementation of nitrogen resulted in a greater enrichment of protein than the addition of the entire nitrogen at the start of the incubation. This effect might be due to better maintenance of the substrate pH, to decreasing inhibition by the nitrogen source, and/or to increasing the efficiency of nitrogen utilization. Heat generation of microbes within the solid substrate can lead to thermal gradients, the result of the limited heat transfer capacity of solid substrates (Mudgett 1986; Saucedo-Castaneda et al. 1990) which could be detrimental to biomass or product formation. At relatively high temperatures (40 to 45”C), protein yield can be severely inhibited (45°C) (Gonzalez-Blanc0 et al. 1990). In the column reactor, the highest temperatures, 40 and 42”C, were found in the bottom and central layers, respectively. High temperatures did not favour the growth of fungi. In a l-1 static bioreactor packed with wet cassava meal and inoculated with A. niger, the temperature gradient in the axial direction from 5 to 35 cm was 0.17’C cm-‘, while
World ]ouml
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S.-S. Yang et al. the maximal temperature gradient in the radial direction, 15 cm from the reactor top, was nearly 30 times greater, at about 5°C err-’ (Saucedo-Castaneda et al. 1990). The same phenomenon was also found in this study (Figure I). A total heat generation in the range of 419 to 2617 J g-’ solids has been reported for the koji process for biomass, enzyme or organic acid production (Aidoo et al. 1982) and 13,398 J g-i substrate has been found in a composting system (Finger et al. 1976). Heat first has to be transferred from the solid substrate to the gaseous phase, by conduction in static incubation, and then has to diffuse through the bulk of the gas to the surroundings (Finger ef al. 1976; Saucedo-Castaneda et al. 1990). Our results with the column reactor indicated that thermal gradients were formed to some extent but also, more importantly, that the temperature in the solids greatly exceeded the ambient temperature. In scaling up such a reactor these problems, particularly the thermal gradients, would be expected to become worse. Filamentous fungi proved to be much better at enriching the protein content of the sweet potato residue than the yeasts. This was probably mainly because the moulds, through growth of their hyphae, were better able to penetrate and spread through the solid substrate. The use of co-cultures, of moulds, yeasts, or a mould with a yeast, failed to significantly enhance the protein enrichment. A column reactor gave results comparable with those obtained in static flask cultures. Temperature and 0, measurements from such a reactor could be useful in developing automatic temperature regulation of solid-state cultivation.
Acknowledgements The authors thank the National Science Council of the Republic of China for financial support (NSC 79-0409-B00252 and NSC 80-0409-B002-27), as well as the Foundation for Research Development, South Africa.
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Raimbault, M. & Alazard, D. 1980 Culture method to study fungal growth in solid fermentation. European ~ownal of Applied Microbiology and Biotechnology 9, 199-209. Reid, I.D. 1989 Solid-state fermentations for biological delignification. Enzyme Microbiological Technology 11, 786803. Saucedo-Castaneda, G., Gutierrez-Rojas, M., Bacquet, G., Raimbault, M. & Viniegra-Gonzalez, G. 1990 Heat transfer simulation in solid substrate fermentation. Biotechnology und Bioengineering 35,
802-808.
Wang, H.H. 1981 Water absorption characteristics of cellulosic wastes and solid state fermentation. In Proceedings of the Second World Congress of Chemical Engineering, 1981, pp. 297-300. Montreal: Canadian Society for Chemical Engineering. Yang, S.S. 1988 Protein enrichment of sweet potato residue with amylolytic yeasts by solid state fermentation. Biotechnology and Bioengineering 32, 88&890. Yang, S.S. 1992 Utilization of agricultural wastes with solid-state fermentation. In Proceedings of Symposium on Microbial and Engineering Technology in Wasfes Treatment, eds Chang, ST., Wong, M.H., Wong, P.K. & Wong, Y.S. pp. 170-194. Hong Kong: Commercial Press. Yang, S.S., Chang, S.L., Wei, C.B. & Lin, H.C. 1991 Reduction of waste production in the Kjeldahl method. ]ownal of the Biomass Energy Sociefy of China 10, 147-155. Yang, S.S. & Chiu, W.F. 1986 Protease production with sweet potato residue by solid state fermentation, Chinese ~ournul of Microbiology and Immunology 19, 276-288. Yang, S.S. & Yuan, S.S. 1990 Oxytetracycline production by Strepfomyces rimosw in solid state fermentation of sweet potato residue. World ]ournul of Microbiology and Biotechnology 6, 236-244.
(Received 1992)
in revised form
30 October
1992;
accepted
2 November
of Microbiology
and Biotechnology,
9, 256-264
Protein enrichment of sweet potato residue by solid-state cultivation with mono- and co-cultures of amylolytic fungi S.-S. Yang,* H.-D. Jang, C.-M. Liew and J.C. du Preez The degree of protein enrichment of sweet potato residue by different amylolytic moulds in solid-state cultivation was much greater than that obtained using amylolytic yeasts. The optimum initial moisture content for protein enrichment was about 65% (w/w). Adding nitrogen sources to the culture twice (at the start of the incubation and after 24 h) considerably improved the final protein content. A co-culture of amylolytic mycelial fungi yielded a at 30°C. In a column reactor, the highest product with 32% (w/w) crude protein after 4 days ’ incubation temperatures reached were 42°C and 40°C and the minimum 0, concentrations were 1.5% and 2.5% of full saturation in the central and bottom layers, respectively. Key words:
Amylolytic
fungi, co-culture,
protein
enrichment,
In 1990, Taiwan imported 1.99 x IO6 tons of soybeans, costing US$5.29 x 10’. from the USA (Economics and Planning Department 1991). Consequently, the development of local protein resources for animal feed from renewable raw materials is urgently required. The substrates traditionally used in solid-state cultivations for the production of foodstuffs are rice, wheat, millet, barley, maize and soybeans. However, use of agricultural wastes such as sweet potato residue, which is not naturally a good protein source for animal feed, might also be practicable in Taiwan because of their abundant supply and reasonable cost. Sweet potato starch is readily converted to biomass by many microorganisms capable of rapid growth (Yang & Chiu 1986; Yang 1988; Yang & Yuan 1990). In order to be economically viable, it must be possible to perform the bioconversion, of starchy materials into protein, at the rural level. Solid-state cultivation facilitates this by reducing the cost of microbial cultivation, by improving the in vitro rumen digestibility of the substrate, and by increasing the protein and fat contents of starchy or cellulosic materials (Grant et al. 1978; Raimbault & Alazard 1980; Yang 1988). We previously reported that
solid-state
cultivation.
sweet potato residue could be enriched from an initial value of 6% up to 21% (w/w) crude protein within 3 days by solid-state cultivation with amylolytic yeasts (Yang 1988). In this article, we report protein enrichment of sweet potato residue by solid-state cultivation with mono- and co-cultures of amylolytic moulds and yeasts, using static flask cultures as well as a column reactor.
Materials
and Methods
Sweet Potato Residue Sweet potato residue
was purchased from a local market in Taiwan and passed through a sieve (four to 16 mesh) to remove dust and large aggregates. The residue contained (w/w): 14.0 to 16.1% moisture; 2.32 to 3.11% crude protein; 2.7 to 3.6% ash; 16.1 to 18.0% crude fibre; and 65.4 to 70.0% carbohydrate (Yang 1988). Microorganisms Saccharomyces
cerevisiae Y-187, Y-188 and Y-191 were provided by C. F. Lin (Microbial Resources Institute, Taiwan). Saccharomyces diastaticLts IF0 1015 STA 1 (D), IF0 1046 STA 1, STA 2 (D) and Saccharomyces sp. IF0 1426 (D) were obtained from W. H. Wang (Research Institute for Wines, Taiwan Tobacco and Wine Monopoly
Bureau).
Schwanniomyces
occidentalis
853 was from
the
Department of Microbiology and Biochemistry, University of the Orange Free State, Bloemfontein, South Africa. Yarrowia (Candida] S.-S. Yang, H.-D. Jang and C.-M. Liew are with the Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan 10617, Republic of China; fax: 666 2 3633123. J.C. du Preez is with the Department of Microbiology and Biochemistry, University of the Orange Free State, Bloemfontein 9300, South Africa. * Corresponding author.
@ 1993 Rapid Communications
258
World Journal
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of Oxford
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Microbiology and Biotechnology. Vol 9, 1993
Aspergillus niger Tainan, Mucor sp. NTLJ-AM-364 and Rhizoptrs sp. NRRL-688 and NRRL-695 were from the Department of Agricultural Chemistry, National Taiwan University, Taipei. All the microorganisms used had amylolytic activity.
lipolytica,
Protein C&m The
Media
and Cultlrre
spores or cells ml-’ with sterile water. For co-cultures a I:1 ratio of cells or spores was used. The solid substrate usually comprised (g): sweet potato residue, 100; (NH,),SO,, 1.25; urea, 1.25; and KH,PO,, 1.0. In some cases, different concentrations of the nitrogen sources were used (see Results). This substrate was distributed to Erlenmeyer flasks, autoclaved, mixed thoroughly with the spores or cells and the required amount of sterile water and incubated statically at 30°C for 3 to 9 days, with mixing once daily by rotating each flask by hand. The depth of medium in each flask was about 2 cm. The pH of the substrate was measured directly by immersing the electrode into the substrate, or determined after mixing an aliquot with five times its volume of distilled water. Coium~
Each sample for 20 min. directly by total nitrogen determined.
and Moisture
The
Fungal
Saccharomyces
1426 1046
of protein
amylolytic
for
further
fungi
enrichment
obtained in Table
study.
contents and pH of sweet for 4 days at 30°C.’
potato
residue
after
the growth
of different
Protein content (% w/w)
PH
Initial
Final
Initial
Final
Initial
Final
66 66
69 68
5.2 5.8
3.8 3.9
3.5
a.5
4.0 3.0
a.0 5.0
diastaticus
IF0
Sacc. Sacc.
cerevisiae cerevisiae
Y-187 Y-l 91
67 68
68 68
4.9 4.5
4.5 4.0
B53
66 66
74 76
5.6 5.3
3.0 3.2
3.0 3.5 4.0
5.5 10.0 11.0
67
78
4.6
3.7
3.5
la.0
66 65 66
72 71 69
4.7 4.5 4.2
4.1 4.1 4.0
3.5 3.5 3.0
15.0 16.5 13.5
A. niger Rhizopus Rhizopus Rhizopus * values per
Tainan sp. NRRL-688 sp. NRRL-695 sp. TBR are the means
g substrate
(dry
of triplicate wt).
experiments,
the
I. With
Sacc.
Schw. occidentalis Y. lipolytica
with
Rhizopcls and TBR, and A. niger Tainan,
is shown
Effect of Initial Moisture Content In all experiments, the moisture content of the substrate increased, the pH value decreased by varying degrees and protein enrichment was affected by initial moisture content. For example, with Saccharomyces sp. IF0 1426, the increase in moisture content was between 0.5% and 3.1% of total weight after 5 days (Table 2), whereas final protein content gradually increased with increasing initial moisture content up to 66.8% initial moisture. When the cultivation lasted more than 5 days, the protein content of the final product decreased slightly (data not shown). With Rhizopus sp.
Moisture content (% w/w)
sp. IF0
Organisms
sp. NRRL-688, NRRL-695 the protein content of the biomass was increased from an initial value of about 3% to 18% (w/w). Schw. occidenfalis B53 and Y. kpolytica gave final protein contents of 10% to ll%, and Sacc. cereuisiae Y-187 and Y-191, Sacc. diastaticus IF0 1046 and Saccharomyces sp. IF0 1426 gave 5% to 9%. In the light of these results, Saccharomyces sp. IF0 1426, Schw. occidentalis B53, Y. lipolyfica, A. niger Tainan and Rhizopus sp. NRRL-688, NRRL-695 and TBR were selected
Contents
strain
degree
different
was extracted with five vol distilled water by shaking and the soluble nitrogen in the extract was determined the Kjeldahl method (Meloan & Pomeranz 1980). The content of each sample, prior to extraction, was also The crude protein content was calculated from the
Table 1. The protein and moisture fungi in static solid-state culture
of Test
Selection
Reacior
Ash
potato residue
Results
The home-made, plastic column reactor (14.7 cm inner diam, 15.0 cm outer diam, 50.0 cm height) had equi-distant sampling holes, 10 cm apart along the column length, which were fitted with thermocouples and gas-sampling tubes. The temperature was measured with al-type thermocouple and digital thermometer. The 0, and CO, contents of the solid substrate were assayed by aspiration with a gas-tight syringe, sealed with a rubber bung, and analysis by gas chromatography using a thermal conductivity detector (Shimadzu GC-6A, Japan). Normal laboratory air was used as the control. No forced aeration was used. The solids were mixed by manually turning the column once daily.
Nitrogen, Protein,
of sweet
difference between the total nitrogen and soluble nitrogen contents, using the conversion factor of 6.25 (Yang et al. 1991). The ash content of samples was determined gravimetrically after 16 to 20 h at 550 to 600°C (Horwitz 1980). The moisture content of the culture was determined by drying a sample at 6O’C under vacuum for 8 to I2 h to constant mass (Yang 1988).
Conditions
amylolytic yeasts were cultivated at 30°C on yeast/malt extract/agar slants, containing (g I-‘): soluble starch, 10; yeast extract, 3; malt extract, 3; peptone, 5; and agar, 20. Moulds were cultivated at 30°C on potato/dextrose/agar slants, containing (g I-‘): potato infusion, 4; dextrose, 20; and agar, 20. Spores or cells were harvested with a Tween 80 solution (5 ml, 0.05% v/v) which was then adjusted to give lo7 to 10’
Solid-sfafe
enrichment
each
using
an inoculum
of IO’ to IO* spores
or cells
S.-S. Yung et al. Table 2. The effect of the residue In static solid-state
initial moisture content culture at 30°C.
on the protein
Parameter
Incubation 0
Saccharomyces sp. I FO 1426 Moisture content (% w/w)
.56
Protein
content
Rhizopus Moisture
(%
w/w)
sp. NRRL-695 content (% w/w)
PH
Protein
content
(% w/w)
56
75 80
75 80
-
81
-
-
-
-
4.5 4.9
-
-
-
-
4.9 4.7
-
-
-
3.2 3.3
3.4 3.7
3.2 3.2
4.9 5.2
3.2 3.2
5.6 5.6
56 60
56
65
60 66
69 76
71 76
80
81
-
-
3.6 5.3
-
6.9 6.5
-
6.4 6.2
-
57 61
57 63
67 71
68 73
76
78 82
81
potato
4
3
60 69 71 77
5.1 5.2
4.5 4.8
4.2 4.2
4.4 4.7
5.1 5.1
5.1 4.0
4.6
4.2 3.9
5.1 5.1
4.6 4.1
3.8 3.7 3.3
3.8 3.6
7.0 6.9
9.9 10.5
3.8 3.8
a.2 9.0 9.6 10.0
-
5
56 61 70 73 78 82
-
4.3 3.8
-
4.0
3.8 3.8 4.0 4.0
5.6 8.4 7.6 7.2 7.1
58 63 69 73 79 83 4.5 4.9 4.6
69 74 78 84 4.8 4.8 4.2
4.3
4.5 4.6 4.6
12.3 12.3
14.3 15.2
17.8 17.8
10.6 10.6 11.1
13.1 13.8 13.1
17.0 17.6
18.0 18.5
11.2
13.0
17.6 17.0
18.6 18.6
3.5 3.5
5.0 4.8
58 64
determined.
NRRL-695, the most rapid initial increase in protein content was found when initial moisture content exceeded 68.8% (Table 2) but by the end of the 5 days’ incubation protein contents were similar, irrespective of initial moisture content.
Nifrogen Supplemenfafion To improve the efficiency of utilization of the nitrogen source, a mixture of ammonium sulphate and urea (at a 1: 1
260
2
-
of sweet
(days)
56 59 67 71
3.3 3.4 -Not
time
59 67 71
4.8 4.6
PH
1
enrichment
World ]oumal of Microbiology and Biofechnology, Vol 9, 1993
w/w ratio) was added to the substrate at a rate of 2% (w/w) both at the start of the incubation (as usual) and again after 24 h. The addition of the nitrogen sources in two increments greatly enhanced the degree of protein enrichment, resulting in a final protein content 53% to 96% higher than in cultures not given extra nitrogen at 24 h. The highest protein contents were obtained with the moulds Rhizopus sp. NRRL-695 (26% w/w) and A. niger Tainan (29% w/w), whereas maximum protein contents obtained with the yeasts were only in the range of 17% to 18% (w/w) (Table 3).
Profein enrichment of sweet potafo residue
Table 3. The effect of nitrogen supplementation on the protein enrichment with different fungi in static flask solid-state culture for 4 days at 30°C. Fungai
strain
Moisture content (% w/w)
Saccharomyces sp. IF0 1426 Control’ Nitrogen supplementationt Sch w. occident&is 053 Control Nitrogen supplementation
of sweet
potato
residue
Protein content (% w/w)
PH
initial
Final
initial
Final
initial
Final
66 64
69 68
5.2 5.5
3.8 5.7
3.5 3.6
9 17
66 64
74 68
5.6 4.7
3.0 3.7
3.3 3.3
10 18
66 64
76 67
5.3 4.4
3.2 7.4
4.1 4.2
11 17
67 63
78 77
4.6 5.4
3.7 6.4
3.4 3.7
16 29
65 64
71 76
4.5 5.5
4.1 a.3
3.3 3.4
16 26
Y. lipolytica Control Nitrogen supplementation A. niger Tainan Control Nitrogen supplementation Rhizopus sp. NRRL-695 Control Nitrogen supplementation * Addition t Addition
of 1% (w/w) of 1% (w/w)
each each
of ammonium of ammonium
suiphate sulphate
Cultivation in a Column Reactor In the column reactor, there were increases in moisture content of between 1% and 4% and 8% and IO% total weight over 4 days with Saccharomyces sp. and A. niger, respectively. The medium became more acidic, changing from an initial pH of 4.3 to 4.4 to a final pH of 3.2 to 3.8. The Saccharomyces strain only gave a final mean protein content of 9% (w/w), whereas A. nip gave 19% (w/w).
Mono- and Co-cultures of Amylolytic Fungi Whereas the A. niger and l&ups sp. co-culture yielded the highest final protein content of the study (32% w/w), which was a slight improvement over the values obtained with these moulds in mono-cultures, the use of yeast co-cultures or a yeast together with a mould failed to improve on the final protein content obtained with the better of the two microorganisms in mono-culture (Table 4). Again, moisture content increased during incubation; the final moisture content was greater with the moulds than with the yeasts. Also as before, the variation in pH was largely dependent on the particular microorganism; some co-cultures became less acidic. For example, growth of Schw. occidentalis caused the pH to decrease to pH 3.6 within 2 days, whereas with Y. lipolytica it increased to pH 7.4, and with the moulds the pH value initially decreased and then increased. In the case of the co-cultures, the pattern of pH change depended on the combination of microorganisms used.
and urea and urea
prior prior
to inoculation. to inoculation
and again
after
24 h.
The ash content, which gives an indication of the amount of organic material lost through microbial metabolism (for example as CO,), increased during incubation; A. niger Tainan had the highest value (followed by the A. n&r Tainan and Rhizopus sp. NRRL-695 co-culture) and the Saccharomyces sp. IF0 1426 and Y. lipolytica co-culture had the lowest value. The observed increases in protein:ash ratios indicated that the increase in the protein content resulted from microbial growth and not only from the loss of organic material from the solids. Temperature and Oxygen ProjIes in &he Solid .%&rate The temperature profile in the solid substrate column reactor with Schw. occidentalis B53 is shown in Figure 1. The highest temperatures of 42°C and 40°C were reached in the central and bottom layers, respectively, after 18 h incubation at a room temperature of 23°C to 30°C. On mixing the solids, the temperatures decreased abruptly, then sharply increased during the subsequent active microbial growth which resulted from the increased 0, supply and fresh substrate contact. The temperatures of the central layer usually slightly exceeded those of the lower layer. The concentration of 0, in the solid substrate decreased gradually during incubation (Figure 2). Each time the column reactor was turned to mix the contents, the 0, concentration increased sharply, then decreased rapidly as vigorous microbial respiration proceeded. The lowest 0, concentrations reached were 1.5% and 2.5% in the bottom and central layers, respectively.
World Jamal
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S.-S. Yang et al.
Table 4. Protein enrichment of sweet potato residue cultivation at 30°C with nitrogen supplementation.” Fungal
Flask
strain
with mono-
Culture period (days)
and co-cultures
Moisture content (% w/w)
fungi
Schw.
sp. IF0
occidentalis
1426
853
PH
Ash (% w/w)
4.6 4.8
4 12
5.7 -
17 3
0
64
5.5
2 4
64
6.0 5.7
68 64
0 2
Tainan
Rhizopus
Protein content (% w/w)
Column
13 18
-
4 12
6.8 7.4
-
5.4 4.3
4.2 6.9
77 64
6.4 5.5
8.8 4.6
4
73 76
4.8 8.3
6.8 7.2
0
63
5.1
4.3
4
2 4
65
4.9 5.0
4.8
12
5.5 4.6
18 5
5.5 6.0 4.4
12 18 4
4.9 4.6 -
13
67 63 74
0 2 4 0 2
sp. NRRL-695
3.6 3.7 4.4
64 65
2 4
A. niger
4.7
69 68
4 0
Y. lipolytica
17 4 17 29 3 17 26
culture
Saccharomyces
Schw.
sp. IF0
occidentalis
Saccharomyces Y. lipolytica
1426
853
sp. IF0
SC/W. occidentalis Y. lipolytica Saccharomyces and Rhizopus
69 64
0 2 1426
and
853
and
sp. IF0
1426
sp. NRRL-695
4.8 4.5
4 0
89 71 65
2 4
66 66
5.9 7.6
0 2
64 69
4.5 5.1
4 0 2
71 65
6.3 5.5
4.6
70
4
73 64 73
4.3 4.2 4.7
5.9 6.4 -
4.4 5.5
0 2
4.6
-
Y. lipolytica
4 0 2
77 65
8.5 5.3
4.8
71
4
4.5 8.1
0 2
76 64 74
4 0 2 4
and
Rhizopus
sp. NRRL-695
A. niger Rhizopus
Tainan and sp. NRRL-695
Blank
l The culture conditions w/w) at 24 h.
were
as in Table
1, but with
19 4 13
-
Schw. occidentalis B53 and Rhizopus sp. NRRL-695
-Not
in a solid-state
culture
Saccharomyces
19 4 17 26 4 14
-
26 4
6.4
13
5.4 4.6
8.2 4.5 6.9
27 4 16
79 64 65
6.8 4.6 4.4
8.3 4.6 4.7
32 3
64
4.7
4.6
supplementation
with
(NH,),SOI
(1%
w/w)
3 3 and
urea
(1%
determined.
Discussion The water-holding capacity of a sample can be used as an index of aerobiosis (Wang 1981). The initial moisture content of the sweet potato residue used in these solid-state
262
of different
World Journal
of
Microbtology and Bmtechnology, Vof 9. 1993
cultivations was usually within the range of 64% to 68%; i.e. less than its water-holding capacity of 72% (Yang 1988). Therefore, suitable conditions for aerobic microbial growth existed in the cultures. During cultivation, however, the moisture content of the substrate increased substantially,
enrichment of sweet potato residue
Protein
bottom layer
201
^
R
40
20
0
Time Figure 2. of Schw. aspirated from the from the
22 0
20
40 Time (h)
60
80
100
Figure 1. Temperature profiles during the solid state cultivation of Schw. occidentalis 853 in a column reactor. Thermocouples were situated on the central vertical axis (A) or 3.5cm horizontally from it (0, a), either 10 cm from the bottom of the reactor or in the central layer, 20cm from the bottom of the reactor. A--Room temperature.
probably due to the production of metabolic water or the release of water resulting from the oxidation of carbohydrates. This increase has also been observed in mushroom spawn (Wang 1981), protein enrichment of sugar beet pulp (Durand & Chereau 1988), enzyme and antibiotic production from sweet potato residue (Yang 1992; Yang & Yuan 1990), liquor brewing from kuo-Lang (Lai 1984), and koji manufacture from cereals (Narahara et al. 1982). It follows, therefore, that the culture may become O,-limited as the cultivation proceeds, as indicated by Figure 2. That this can retard growth has been demonstrated in cultures of Schw. occidentalis 853, in which OJimitation severely restricted amylase production (Horn 1990) and in the inhibition of the degradation of organic materials in cornposting rubbish (Nakasaki et al. 1990). CO, formation and 0, consumption in solid-state fermentations are probably a function of radius position (Saucedo-Castaneda et al. 1990). Maximal CO, evolution rates were found in an intermediate region of the growth profile of A. tiger during protein enrichment of sugar-cane by-products (GonzalezBlanc0 et al. 1990). In the present study, 0, concentrations decreased as incubation progressed. Mixing or aeration may be necessary, therefore, for efficient protein enrichment by aerobic microbes in solid-state cultivation.
Oxygen
concentration
60
80
100
(h) during
the solid-state cultivation reactor. Gas samples were the centre of the horizontal plane either 1Ocm of the reactor (A) or in the central layer, 20 cm of the reactor (0).
occident&is 853 in a column from bottom bottom
The pH of solid-state cultivations can be controlled by using different ratios of ammonium salts and urea (Mitchell et al. 1988; Yang 1988), ammonium nitrate or sodium nitrate (Yang & Chiu 1986) a buffering agent such as CaCO,, or the salt of an organic acid (Reid 1989; Yang & Yuan 1990). However, our results show that use of a mixture of an ammonium salt and urea was only partially effective and that the degree of change in pH depended on the metabolic activities of the particular microorganism used. The large changes in pH noted in some of our cultivations can severely retard starch hydrolysis and growth. For example, the amylases of Schw. accidentalis B53 are rapidly and irreversibly inactivated below pH 5 (Horn 1990). In order to enrich the protein content of sweet potato residue to 20 to 30% (w/w), the addition of 4% (w/w) of a I: 1 mix of ammonium sulphate and urea was necessary. We found that the incremental supplementation of nitrogen resulted in a greater enrichment of protein than the addition of the entire nitrogen at the start of the incubation. This effect might be due to better maintenance of the substrate pH, to decreasing inhibition by the nitrogen source, and/or to increasing the efficiency of nitrogen utilization. Heat generation of microbes within the solid substrate can lead to thermal gradients, the result of the limited heat transfer capacity of solid substrates (Mudgett 1986; Saucedo-Castaneda et al. 1990) which could be detrimental to biomass or product formation. At relatively high temperatures (40 to 45”C), protein yield can be severely inhibited (45°C) (Gonzalez-Blanc0 et al. 1990). In the column reactor, the highest temperatures, 40 and 42”C, were found in the bottom and central layers, respectively. High temperatures did not favour the growth of fungi. In a l-1 static bioreactor packed with wet cassava meal and inoculated with A. niger, the temperature gradient in the axial direction from 5 to 35 cm was 0.17’C cm-‘, while
World ]ouml
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S.-S. Yang et al. the maximal temperature gradient in the radial direction, 15 cm from the reactor top, was nearly 30 times greater, at about 5°C err-’ (Saucedo-Castaneda et al. 1990). The same phenomenon was also found in this study (Figure I). A total heat generation in the range of 419 to 2617 J g-’ solids has been reported for the koji process for biomass, enzyme or organic acid production (Aidoo et al. 1982) and 13,398 J g-i substrate has been found in a composting system (Finger et al. 1976). Heat first has to be transferred from the solid substrate to the gaseous phase, by conduction in static incubation, and then has to diffuse through the bulk of the gas to the surroundings (Finger ef al. 1976; Saucedo-Castaneda et al. 1990). Our results with the column reactor indicated that thermal gradients were formed to some extent but also, more importantly, that the temperature in the solids greatly exceeded the ambient temperature. In scaling up such a reactor these problems, particularly the thermal gradients, would be expected to become worse. Filamentous fungi proved to be much better at enriching the protein content of the sweet potato residue than the yeasts. This was probably mainly because the moulds, through growth of their hyphae, were better able to penetrate and spread through the solid substrate. The use of co-cultures, of moulds, yeasts, or a mould with a yeast, failed to significantly enhance the protein enrichment. A column reactor gave results comparable with those obtained in static flask cultures. Temperature and 0, measurements from such a reactor could be useful in developing automatic temperature regulation of solid-state cultivation.
Acknowledgements The authors thank the National Science Council of the Republic of China for financial support (NSC 79-0409-B00252 and NSC 80-0409-B002-27), as well as the Foundation for Research Development, South Africa.
References Aidoo,
K.E., Hendry, R. & Wood, B.J.B. 1982 Solid state fermentations. Advances in Applied Microbiology 28, 201-237. Durand, A. & Chereau, D. 1988 A new pilot reactor for solid-state fermentation: application to the protein enrichment of sugar beet pulp. Biotechnology and Bioengineering 31, 476486. Economics and Planning Department 1991 &torn Statistics-1990 Taiwan. Taipei, Taiwan: Economics and Planning Department. Finger, S.M., Hatch, R.T. & Regan, T.M. 1976 Aerobic microbial growth in semisolid matrices: heat and mass transfer limitation. Biotechnology and Bioengineering 18, 1193-1218. Gonzalez-Blanco, P., Saucedo-Castaneda, G. 81 Viniegra-Gonzalez, G. 1990 Protein enrichment of sugar cane by-products using solid-state cultures of Aspergillus terreus ~ownal of Fermenfution and Bioengineering 70, 351-354.
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(Received 1992)
in revised form
30 October
1992;
accepted
2 November
